Marked competitive small molecule for light-activated chemiluminescence detection and light-activated chemiluminescence detection kit

Abstract

The invention relates to a labeled competitive small molecule for light-activated chemiluminescence detection and a light-activated chemiluminescence detection kit, the labeled competitive small molecule has a simple formula of competitive small molecule-marker, the molecular weight of the competitive small molecule is less than 1000Da, the molecular weight of the marker is 1000Da-10000Da, the molecular weight after biotinylation can be improved, the biotinylation efficiency is improved, and the biotinylation efficiency is improved. Subsequent desalination and purification are easy, the purity is high, the impurity content is low, the calibration signal value and the distinction degree are good, and improvement of the precision and the accuracy of clinical detection is facilitated.

Description

Technical Field

[0001] The present application relates to the technical field of biological reagents, and in particular, to a labeled competitive small molecule for photochemiluminescence detection and a photochemiluminescence detection kit. Background Art

[0002] The biotin-avidin system (BAS) is a biological reaction amplification system developed in the 1970s. With the advent of biotin derivatives, BAS has been quickly and widely applied in the field of medical detection. In recent years, a large number of studies have confirmed that the biotin-avidin system can almost bind to various markers that have been successfully studied. The strong binding with high affinity between biotin and avidin and the multi-stage amplification effect make BAS immunolabeling and related tracer analysis more sensitive. It has become a new technology widely used for qualitative, quantitative detection and localization observation research of trace antigens and antibodies.

[0003] In practical applications, for some small molecule hormones, common Biotin-LCLC-NHS reagents are usually used for their biotinylation labeling. Since the molecular weight of small molecule hormones is usually less than 1000 Da, after biotinylation labeling with Biotin-LCLC-NHS, the purity is relatively low and the impurity content is relatively high. Furthermore, when it is applied to chemiluminescence detection, the obtained detection signal value and discrimination degree are both relatively poor, which is not conducive to improving the accuracy and precision of detection. Summary of the Invention

[0004] To solve or partially solve the problems existing in the related technologies, the present application provides a labeled competitive small molecule for photochemiluminescence detection and a photochemiluminescence detection kit, so that after the competitive small molecule is labeled, the purity after purification is relatively high, the impurity content is relatively low, the calibration signal value and discrimination degree are relatively good, which is conducive to improving the accuracy and precision of clinical detection.

[0005] The first aspect of the present application provides a labeled competitive small molecule for photochemiluminescence detection. The simple formula of the labeled competitive small molecule is: competitive small molecule - label, where the molecular weight of the competitive small molecule is less than 1000 Da, and the molecular weight of the label is 1000 Da - 10000 Da.

[0006] According to the labeled competitive small molecule described in the first aspect, the label is a biotinylated long-chain molecule; preferably, the long-chain molecule is a hydrophilic long-chain molecule; more preferably, the hydrophilic long-chain molecule is selected from polyethylene glycol or dextran.

[0007] According to the labeled competitive small molecule described in the first aspect, the polyethylene glycol PEG n is selected from PEG 25 ~PEG200 at least one of; preferably selected from PEG 30 ~PEG 180 at least one of; more preferably selected from PEG 40 ~PEG 150 at least one of; further preferably selected from PEG 70 ~PEG 120 at least one of; and / or

[0008] said dextran [C6H 10 O5] n selected from at least one of dextrans with n = 7 to 55; preferably selected from at least one of dextrans with n = 10 to 50; more preferably selected from at least one of dextrans with n = 16 to 40; further preferably selected from at least one of dextrans with n = 20 to 30.

[0009] The second aspect of the present application provides a photoactivated chemiluminescence detection kit for detecting small molecules, which includes reagent 1 and reagent 2, wherein:

[0010] Reagent 1 includes the labeled competitive small molecule described in any one of the above first aspects;

[0011] Reagent 2 includes luminescent microparticles, and the surface of the luminescent microparticles is coated with an antibody or binding protein of the small molecule to be detected.

[0012] According to the photoactivated chemiluminescence detection kit described in the second aspect, the concentration of reagent 1 is 3 ng / mL to 10 ng / mL, preferably, the concentration of reagent 1 is 5 ng / mL; and / or

[0013] The concentration of reagent 2 is 10 μg / mL to 30 μg / mL, preferably, the concentration of reagent 2 is 20 μg / mL.

[0014] According to the photoactivated chemiluminescence detection kit described in the second aspect, the small molecule to be detected includes hormones, vitamins, etc.; preferably, the small molecule to be detected is sex hormone, thyroid function hormone, adrenal cortical hormone; more preferably, the small molecule to be detected is testosterone, progesterone, estradiol, free triiodothyronine, free thyroxine, aldosterone, etc.

[0015] According to the photoactivated chemiluminescence detection kit described in the second aspect, the small molecule to be detected is detected according to the competitive method, and the competitive small molecule is used as a competitor for the small molecule to be detected; preferably, the competitive small molecule is a derivative or structural analogue of the small molecule to be detected.

[0016] The luminescent immunoassay kit according to the second aspect, wherein the competitive small molecule and the small molecule to be detected can be recognized and bound by the same antibody coated on the surface of the luminescent particles; preferably, the binding affinities of the competitive small molecule and the small molecule to be detected to the antibody are different; more preferably, the binding affinity of the competitive small molecule to the antibody is lower than that of the small molecule to be detected to the antibody.

[0017] The luminescent immunoassay kit according to the second aspect further comprises Reagent 3, which includes a releasing agent, and the releasing agent is a strong acid, a strong base and / or a displacing agent; preferably, the releasing agent is a citrate buffer solution; and / or further comprises Reagent 4, which includes photosensitive particles coated with avidin; preferably, the concentration of Reagent 4 is 40 μg / mL to 50 μg / mL.

[0018] The third aspect of the present application provides an application of the luminescent immunoassay kit according to any one of the above second aspects in detecting a small molecule to be detected; preferably, the molecular weight of the small molecule to be detected is less than 1000 Da.

[0019] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. Detailed Description of the Invention

[0020] To make the present invention easy to understand, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing the specific embodiments and do not represent any limitation.

[0021] When a numerical range is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other specified or intermediate value in the specified range is included in the present invention. The upper and lower limits of these smaller ranges can be independently included in the smaller ranges and are also included in the present invention, subject to any explicit exclusions in the specified range. When the specified range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the present invention.

[0022] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0023] Term Explanation

[0024] As used herein, the term "photosensitive particle" refers to a particle containing a sensitizer that can be activated by energy or an active compound and can generate active intermediates such as reactive oxygen species that can react with luminescent particles. In some specific embodiments of the present application, the photosensitive particle is a polymer particle filled with a photosensitizer, and the photosensitizer can be a photosensitizer known in the art, preferably a compound that is relatively light-stable and does not effectively react with singlet oxygen, such as compounds like methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll, and derivatives of these compounds having 1-50 atom substituents, which are used to make these compounds more lipophilic or more hydrophilic and / or as a linking group for attachment to a specific binding partner. Examples of other photosensitizers known to those skilled in the art can also be used in the present application.

[0025] As used herein, the term "luminescent particle" refers to a particle containing a compound that can react with reactive oxygen species to generate a detectable signal. The photosensitive particle is induced to be activated by energy or an active compound and releases reactive oxygen species in a high-energy state, and the reactive oxygen species in the high-energy state are captured by the nearby luminescent particles, thereby transferring energy to activate the luminescent particles. In some specific embodiments of the present application, the luminescent particle comprises a luminescent composition and a carrier, and the luminescent composition is filled in the carrier and / or coated on the surface of the carrier.

[0026] The present application provides a labeled competitive small molecule for photochemiluminescence detection, and the simplified formula of the labeled competitive small molecule is: competitive small molecule - label, wherein the molecular weight of the competitive small molecule is less than 1000 Da, and the molecular weight of the label is 1000 Da - 10000 Da. By using a label with a molecular weight within a specific range to label the competitive small molecule, the present application can increase the molecular weight of the competitive small molecule after labeling, making desalting and purification easier, obtaining a competitive small molecule reagent with higher purity and less impurity content, and then having better calibration signal values and discrimination degrees when applied to detection, which is beneficial to improving the accuracy and precision of clinical detection.

[0027] It should be noted that the commonly used biotinylation reagent in the prior art is usually Biotin-LCLC-NHS (N-succinimidyl 6-biotinamidohexanoic acid) reagent. After biotinylating the competitive small molecule with Biotin-LCLC-NHS reagent, the molecular weight of the labeled molecule is still relatively small, usually less than 1000 Da. Subsequently, the desalting difficulty is relatively high, which results in a relatively large amount of small molecule impurities and low purity after being labeled with Biotin-LCLC-NHS reagent. Both the detection signal value and the discrimination degree are relatively poor, which is not conducive to improving the accuracy and precision of detection. In this application, by using a high molecular weight label to label the competitive small molecule, the molecular weight is relatively high, the purification is relatively easy, the purity is high, the impurity content is small, the calibration signal value and the discrimination degree are good, which is conducive to improving the accuracy and precision of clinical detection.

[0028] In some embodiments, the label is a biotinylated long-chain molecule. Preferably, the long-chain molecule is a hydrophilic long-chain molecule; more preferably, the hydrophilic long-chain molecule is selected from polyethylene glycol or dextran. That is to say, the label can be a biotinylated hydrophilic long-chain molecule; more preferably, the label can be biotinylated polyethylene glycol or biotinylated dextran. Of course, the hydrophilic long-chain molecule can also be other hydrophilic molecules with a molecular weight within a specified range. Here, only examples are given for illustration and no limitation is made.

[0029] Correspondingly, the molecular formula of the competitive small molecule after being labeled by the corresponding label can be competitive small molecule - polyethylene glycol - biotin, that is, competitive small molecule - PEG n -Biotin, where n represents the number of PEG monomers; or competitive small molecule - dextran - biotin, that is, competitive small molecule - 10 [C6H n O5] 10 -Biotin, where n represents the number of C6H

[0030] In some embodiments, when the label is selected from biotinylated polyethylene glycol PEG n when, the polyethylene glycol PEG n is selected from at least one of PEG 25 ~PEG 200 ; preferably, it is selected from at least one of PEG 30 ~PEG 180 ; more preferably, it is selected from at least one of PEG 40 ~PEG 150 ; further preferably, it is selected from at least one of PEG 70 ~PEG 120 . That is to say, the label is selected from biotinylated PEG 25 ~PEG 200A mixture of at least one or more of them, such that the molecular weight of the marker is between 1000 Da and 10000 Da.

[0031] In some embodiments, when the marker is selected from biotinylated dextran [C6H 10 O5] n when, dextran [C6H 10 O5] n is selected from at least one of dextrans with n = 7 to 55; preferably, at least one of dextrans with n = 10 to 50; more preferably, at least one of dextrans with n = 16 to 40; still more preferably, at least one of dextrans with n = 20 to 30. That is to say, the marker is selected from at least one or a mixture of more than one of biotinylated [C6H 10 O5]7 to [C6H 10 O5] 55 such that the molecular weight of the marker is between 1000 Da and 10000 Da.

[0032] In some embodiments, according to the types of competitive small molecules, different markers can be selected. For example, when the competitive small molecule is a derivative of testosterone, the marker can be biotinylated polyethylene glycol PEG n . When the competitive small molecule is a derivative of estradiol, the marker can be biotinylated dextran [C6H 10 O5] n . Of course, this is only an example here and is not limiting.

[0033] In order to rapidly prepare labeled competitive small molecules, in some embodiments, the competitive small molecule is pre-mixed with an activator in a certain proportion; after obtaining the activated competitive small molecule, it is then reacted with the marker reagent, and after desalting and purification, the labeled competitive small molecule is prepared. Preferably, the activator can be EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide). Preferably, the liquid after the reaction of the activated competitive small molecule with the marker reagent can be desalted and purified according to a desalting column. It can be understood that the molecular weight of the marker is 1000 Da to 10000 D, the retention efficiency of the desalting column for salts and small molecules (<1000 Da) is ≥95%, and by using a high molecular weight marker to label the competitive small molecule, the molecular weight of the labeled competitive small molecule is greater than 1000 Da, and then the molecular weight after biotinylation can be increased. After desalting and purification, a competitive small molecule labeling reagent with higher purity and less impurity content can be more easily obtained, which is beneficial to improving the accuracy and precision of clinical detection.

[0034] The present application also provides a photochemiluminescence detection kit for detecting small molecules, including Reagent 1 and Reagent 2. Among them, Reagent 1 includes the labeled competitive small molecules in any of the above embodiments; Reagent 2 includes luminescent microparticles, and the surface of the luminescent microparticles is coated with an antibody or binding protein of the small molecule to be detected.

[0035] The photochemiluminescence detection kit of the present application is used to detect the target small molecule. In some embodiments, the target small molecule to be detected is detected according to the competitive method, and the labeled competitive small molecule is used as a competitor for the target small molecule to be detected. In some embodiments, the target small molecule to be detected is a hormone or a vitamin; in some preferred embodiments, the target small molecule to be detected is a sex hormone, a thyroid function hormone, or an adrenal cortical hormone; in some more preferred embodiments, the target small molecule to be detected is testosterone, progesterone, estradiol, free triiodothyronine, free thyroxine, aldosterone, etc.

[0036] Furthermore, when performing photochemiluminescence detection by the competitive method, the labeled competitive small molecule is used as a competitor for the target small molecule to be detected, and the competitive small molecule and the target small molecule to be detected can be recognized and bound by the same antibody coated on the surface of the luminescent microparticles. Among them, the antibody coated on the surface of the luminescent microparticles is an antibody of the target small molecule to be detected. For example, the antibody is an antibody of a hormone; for another example, the antibody is an antibody of a sex hormone; for another example, the antibody is a testosterone antibody, a progesterone antibody, or an estradiol antibody.

[0037] Correspondingly, in order to enable both the competitive small molecule in Reagent 1 and the target small molecule to be detected to be recognized and bound by the same antibody, in some embodiments, according to the type of the target small molecule to be detected, a corresponding derivative or structural analog is selected as the competitive small molecule in Reagent 1. For example, when the target small molecule to be detected is a sex hormone, correspondingly, in some specific embodiments, the competitive small molecule is a sex hormone derivative; for example, the competitive small molecule is a testosterone derivative, a progesterone derivative, or an estradiol derivative. The labeled competitive small molecule in Reagent 1 is the labeled testosterone derivative, the labeled progesterone derivative, or the labeled estradiol derivative.

[0038] It can be understood that for the photochemiluminescence detection kit of the present application, according to the different target small molecules to be detected, the competitive small molecules in Reagent 1 are selected accordingly, and according to the type of the competitive small molecule, a corresponding label is selected. For example, when the competitive small molecule is a testosterone derivative, the label can be biotinylated polyethylene glycol, and the simplified formula of the labeled competitive small molecule can be testosterone derivative-PEG n -Biotin. For another example, when the competitive small molecule is an estradiol derivative, the label can be biotinylated dextran [C6H 10 O5] n , and the simplified formula of the labeled competitive small molecule can be estradiol derivative-[C6H 10 O5]n - Biotin.

[0039] In some embodiments, the binding affinities of the competing small molecule and the small molecule to be detected for the antibody are different. More preferably, the binding affinity of the competing small molecule for the antibody is lower than that of the small molecule to be detected for the antibody. By selecting a derivative or structural analogue of the small molecule to be detected as the competing small molecule in Reagent 1, both the labeled competing small molecule and the small molecule to be detected can compete for binding to the same antibody, but with different binding affinities, and thus an obvious competitive effect can be achieved, making the detection results have a clearer discrimination.

[0040] To obtain more accurate detection results, in some embodiments, the concentration of Reagent 1 can be 3 ng / mL to 10 ng / mL. Preferably, the concentration of Reagent 1 can be 4 ng / mL to 8 ng / mL. More preferably, the concentration of Reagent 1 can be, for example, 5 ng / mL, 6 ng / mL, 7 ng / mL, etc. Preferably, Reagent 1 further includes a buffer solution. For example, the buffer solution can be a HEPES buffer solution, etc. Among them, Reagent 1 can be diluted to the required concentration by the buffer solution.

[0041] In some embodiments, the concentration of Reagent 2 can be 10 μg / mL to 30 μg / mL. Preferably, the concentration of Reagent 2 can be 15 μg / mL to 25 μg / mL. More preferably, the concentration of Reagent 2 can be, for example, 15 μg / mL, 18 μg / mL, 23 μg / mL, 25 μg / mL, 30 μg / mL, etc. Preferably, Reagent 2 further includes a buffer solution. For example, the buffer solution can be a Tris buffer solution, etc. Among them, Reagent 2 can be diluted to the required concentration by the buffer solution.

[0042] In some embodiments, the photochemiluminescence detection kit further includes Reagent 3, and Reagent 3 includes a releasing agent, and the releasing agent is a strong acid, a strong base, and / or a displacing agent. Preferably, the releasing agent is a citrate buffer solution. Specifically, taking the small molecule to be detected as a sex hormone as an example, the releasing agent in the present application refers to a chemical reagent that can release the small molecule to be detected from sex hormone-binding globulin or albumin. In some embodiments, the concentration of Reagent 3 can be 0.1 mol / L to 0.5 mol / L. Preferably, it can be, for example, 0.1 mol / L, 0.2 mol / L, 0.4 mol / L, 0.5 mol / L, etc. This is only for illustration and not for limitation.

[0043] In some embodiments, the photochemiluminescence detection kit further includes Reagent 4, and Reagent 4 includes avidin-coated photosensitive microparticles. Preferably, the concentration of Reagent 4 can be 40 μg / mL to 50 μg / mL. More preferably, the concentration of Reagent 4 can be, for example, 50 μg / mL. Among them, Reagent 4 can be used as a general reagent and is applicable to photochemiluminescence detection kits for detecting different target small molecules.

[0044] In summary, according to the above photochemiluminescence detection kit, by using a competitive small molecule-label to perform chemiluminescence detection on the target small molecule to be detected according to the competitive method, it has better detection signal values and discrimination.

[0045] The present application also provides an application of a photochemiluminescence detection kit in detecting a target small molecule to be detected. For example, a method for detecting a target small molecule to be detected according to the above photochemiluminescence detection kit. Among them, the molecular weight of the target small molecule to be detected is less than 1000 Da.

[0046] In some embodiments, the detection method includes: mixing the sample to be detected, Reagent 1, Reagent 2, Reagent 3, and Reagent 4 in a preset manner for reaction, then irradiating with excitation light, measuring the amount of emitted light photons to obtain a light signal value, and quantitatively or qualitatively obtaining the detection result of the target small molecule to be detected in the sample to be detected according to the light signal value.

[0047] In a specific embodiment, the sample to be detected, Reagent 1, Reagent 2, and Reagent 3 are mixed and incubated, and then Reagent 4 is added for mixing and incubation. For example, the sample to be detected, Reagent 1, Reagent 2, and Reagent 3 are mixed and incubated at 37 °C for 15 min, then an appropriate amount of Reagent 4 is added, and after incubation at 37 °C for 10 min, it is irradiated with the excitation light of the photochemiluminescence detection system to measure the light signal value.

[0048] The method of the present application is detected according to the test principle of the competitive method, and is used for quantitatively or qualitatively detecting the target small molecule in the sample to be detected. The operation is simple and convenient, and the competitive small molecule-label has good signal values and discrimination, and more accurate detection results can be obtained.

[0049] To make the present invention easier to understand, the following will further illustrate the present invention in detail with reference to embodiments. These embodiments are only illustrative and are not limited to the application scope of the present invention. The raw materials or components used in the present invention can be obtained through commercial channels or conventional methods without special instructions.

[0050] Below, taking the derivative of sex hormone testosterone T, abbreviated as T derivative, as an example of the competitive small molecule, Biotin-PEG with molecular weights of 588 Da, 1000 Da, 5000 Da, 10 kDa, and 20 kDa is used for the T derivative respectively. nLabel with -NH2 reagent to obtain T derivative - PEG n -Biotin. Then purify with a desalting column and finally detect the signal value and discrimination on the amplified luminescent proximity homogeneous assay platform. Biotin-PEG n -NH2 reagent is biotin polyethylene glycol active ester reagent, hereinafter referred to as Biotin-PEG n -NH2.

[0051] Example 1: Preparation of T derivative - PEG n -Biotin

[0052] 1.1 The main experimental raw materials and equipment are shown in Table 1:

[0053] Table 1

[0054]

[0055]

[0056] 1.2 Take a centrifuge tube and weigh 0.2 mg of T derivative and dissolve it in 200 μL of DMSO solution to prepare a T derivative reagent. Weigh 5 mg of EDC and dissolve it in 500 μL of pure water to prepare an EDC reagent. Take 5 μL of the above EDC reagent and add it to the prepared T derivative reagent, mix well to obtain an activated T derivative reagent.

[0057] 1.3 Dissolve 0.26 μmol of Biotin-PEG n -NH2 (588 Da), Biotin-PEG n -NH2 (1K Da), Biotin-PEG n -NH2 (5K Da), Biotin-PEG n -NH2 (10K Da) and Biotin-PEG n -NH2 (20K Da) in DMSO solution respectively to prepare 5 kinds of Biotin-PEG n -NH2 reagents with different molecular weights. Mix each Biotin-PEG n -NH2 reagent with the activated T derivative reagent respectively and let it stand and react at 2 °C - 8 °C for 18 h to obtain the corresponding reaction solution.

[0058] 1.4 Desalting and purification process:

[0059] Desalt and purify the above 5 kinds of reaction solutions according to the following steps.

[0060] First centrifugation: Take a Zeba desalting column, remove the tail of the desalting column, and loosen the lid of the desalting column. Place it in a suitable centrifuge tube and weigh it on an electronic balance for balance. Centrifuge at 2°C - 8°C (set at 4°C), 1500g, for 2 minutes.

[0061] Second centrifugation: Replace with a new centrifuge tube, add 0.1M NaHCO3 (pH 8.5) buffer solution to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2°C - 8°C (set at 4°C), 1500g, for 2 minutes.

[0062] Third centrifugation: Replace with a new centrifuge tube, add 0.1M NaHCO3 (pH 8.5) buffer solution to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2°C - 8°C (set at 4°C), 1500g, for 2 minutes. Recover the liquid in the centrifuge tube.

[0063] Fourth centrifugation: Replace with a new centrifuge tube, add T derivative - PEG n -Biotin to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2°C - 8°C (set at 4°C), 1500g, for 2 minutes. The liquid collected in the centrifuge tube is the purified T derivative - PEG n -Biotin reagent, which is the T derivative labeled with biotin polyethylene glycol active ester.

[0064] Example 2: Preparation of a photochemiluminescence immunoassay kit for detecting testosterone (T)

[0065] 1. Dilute the T derivative - PEG n -Biotin reagents with different molecular weights prepared in Example 1 above to 5 ng / mL with HEPES buffer solution to obtain 5 corresponding reagent 1.

[0066] 2. Dilute the luminescent microparticles coated with testosterone antibody to 20 μg / mL with Tris buffer solution to obtain reagent 2.

[0067] 3. Prepare 0.2 mol / L citrate buffer solution, dispense it, and obtain reagent 3.

[0068] 4. Assemble reagent 1, reagent 2, and reagent 3 into a corresponding set of complete reagents respectively to obtain 5 sets of complete reagents.

[0069] 5. Prepare a photosensitive microparticle solution with a concentration of 50 μg / mL in advance as a general reagent, namely reagent 4.

[0070] Example 3: Detection of testosterone (T)

[0071] Prepare 6 different concentrations of testosterone reagents as calibration products, and mix the 5 sets of complete reagents prepared in Example 2 above with the sample to be tested Detection was carried out on a 500 photochemiluminescence detection system.

[0072] 1. Mix 20 μL of the sample to be tested, 25 μL of Reagent 1, 25 μL of Reagent 2, and 20 μL of Reagent 3 evenly, and incubate at 37 °C for 15 min.

[0073] 2. Add 175 μL of the universal reagent Reagent 4, and incubate at 37 °C for 10 min.

[0074] 3. After the photoexcitation reaction, read the light signal values of each group of tests respectively. The experimental data are shown in Table 2 below.

[0075] Table 2

[0076]

[0077] It can be seen from the data in Table 2 that when the molecular weight of the Biotin-PEG n -NH2 reagent is 588 Da, the corresponding calibrator signal values are generally relatively low, and the overall discrimination of the reagent is 10.2; when the molecular weight of the Biotin-PEG n -NH2 reagent increases from 588 Da to 1 kDa, based on the data corresponding to 588 Da, the signal values corresponding to different calibrator concentrations increase by an average of 7.8 times, and the inhibition rate at the highest concentration increases by 1.8 times; the overall discrimination of the reagent is 18.2. When the molecular weight of the Biotin-PEG n -NH2 reagent continues to increase from 1 kDa to 5 kDa, based on the 588 Da data, the signal values increase by an average of 10 times, the inhibition rate at the highest concentration increases by 5.2 times, and the overall discrimination of the reagent is 53.1. When the molecular weight of the Biotin-PEG n -NH2 reagent continues to increase from 5 kDa to 10 kDa, based on the 588 Da data, the signal values increase by an average of 7.8 times, the inhibition rate at the highest concentration increases by 1.8 times, and the overall discrimination of the reagent is 18.8. When the molecular weight of the Biotin-PEG n -NH2 reagent continues to increase from 10 kDa to 20 kDa, based on the 588 Da data, the signal values increase by an average of 5.9 times, the inhibition rate at the highest concentration increases by 1.3 times, and the overall discrimination of the reagent is 13.3.

[0078] It should be noted that the theoretical values of the molecular weights of the five Biotin-PEG n -NH2 are 948 Da, 1360 Da, 5360 Da, 10360 Da, and 20360 Da respectively, and the molecular weight of the T derivative is 360. The retention efficiency of the desalting column for salts and small molecules (molecular weight < 1000 Da) is ≥ 95%. When the T derivative-PEG n-When the molecular weight of Biotin is around 1000Da, it will also be retained in the desalting column with a high probability, resulting in the T derivative-PEG in the obtained reagent n -Biotin content decreases, and the signal value decreases. n When the molecular weight of -NH2 reagent reaches 5K Da, T derivative and T derivative-PEG can be fully separated n -Biotin, the effective reagent content is high, and the signal value is correspondingly improved. However, the separation effect will not increase significantly if the molecular weight continues to increase. On the contrary, after exceeding 10K Da, the reaction efficiency is affected due to the large molecular chain length, resulting in a decrease in signal value and poor discrimination.

[0079] In summary, Biotin-PEG n -NH2 has a better effect when its molecular weight is between 1KDa and 10KDa, and it has the best effect on labeling small molecule hormones when its molecular weight is 5K Da.

[0080] Example 4: Detection of estradiol (E2)

[0081] Referring to the method of Example 1, the E2 derivative was labeled with 3K Da and 10K Da biotin-dextran reagents to obtain the desalted and purified E2 derivative - [C6H 10 O5] n -Biotin reagent.

[0082] Referring to the method of Example 2, a photochemiluminescence detection kit for detecting sex hormone E2 was prepared.

[0083] In this example, 6 sets of E2 reagents with different concentrations were prepared as calibrants, and the two sets of complete reagents prepared in Example 2 were mixed with the samples to be tested. Detection was performed on a 500 photochemiluminescence detection system.

[0084] The detection process of this embodiment refers to Example 3, and the experimental data are shown in Table 3 below.

[0085] Table 3

[0086]

[0087] This example shows that the larger the molecular weight of the marker, the better. The biotin-dextran reagent with a molecular weight of 3000 Da has the best labeling effect.

[0088] When the labeled competitive small molecule provided by the present application is used in a luminescent oxygen channeling immunoassay (LOCI) kit, due to its good purity, the detection signal value and discrimination are relatively good, and both the detection signal value and discrimination on the LOCI platform are better than those of other reagents, such as the small molecule biotinylated by Biotin-PEG-NH2 reagent.

[0089] The embodiments of the present application have been described above. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of the terms used herein is intended to best explain the principles of the embodiments, the practical application, or the improvement of the technology in the market, or to enable other ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A labeled competitive small molecule for photochemiluminescence detection, characterized in that, The simplified formula of the labeled competitive small molecule is: competitive small molecule - label, where the molecular weight of the competitive small molecule is less than 1000 Da, and the molecular weight of the label is 1000 Da - 10000 Da.

2. The labeled competitive small molecule according to claim 1, wherein The label is a biotinylated long-chain molecule; preferably, the long-chain molecule is a hydrophilic long-chain molecule; more preferably, the hydrophilic long-chain molecule is selected from polyethylene glycol or dextran.

3. The labeled competitive small molecule according to claim 2, wherein The polyethylene glycol PEG n is selected from PEG 25 to PEG 200 and is at least one of them; preferably, it is selected from PEG 30 to PEG 180 and is at least one of them; more preferably, it is selected from PEG 40 to PEG 150 and is at least one of them; further preferably, it is selected from PEG 70 to PEG 120 and is at least one of them; and / or The dextran [C6H 10 O5] n is at least one selected from dextrans with n = 7 to 55; preferably, at least one selected from dextrans with n = 10 to 50; more preferably, at least one selected from dextrans with n = 16 to 40; still more preferably, at least one selected from dextrans with n = 20 to 30.

4. A photochemiluminescence detection kit for detecting small molecules, characterized in that, It includes reagent 1 and reagent 2, where: Reagent 1 includes the labeled competitive small molecule as described in any one of claims 1 to 3; Reagent 2 includes luminescent microparticles, and the surface of the luminescent microparticles is coated with an antibody or binding protein of the small molecule to be detected.

5. The chemiluminescence immunoassay kit according to claim 4, wherein: The concentration of reagent 1 is 3 ng / mL - 10 ng / mL, preferably, the concentration of reagent 1 is 5 ng / mL; and / or The concentration of reagent 2 is 10 μg / mL - 30 μg / mL, preferably, the concentration of reagent 2 is 20 μg / mL.

6. The chemiluminescence immunoassay kit according to claim 4, wherein: The small molecule to be detected includes hormones, vitamins, etc.; preferably, the small molecule to be detected is sex hormone, thyroid function hormone, adrenal cortical hormone; more preferably, the small molecule to be detected is testosterone, progesterone, estradiol, free triiodothyronine, free thyroxine, aldosterone, etc.

7. The chemiluminescence immunoassay kit according to claim 4 or 6, wherein: The small molecule to be detected is detected according to the competitive method, and the competitive small molecule is used as a competitor for the small molecule to be detected; preferably, the competitive small molecule is a derivative or structural analogue of the small molecule to be detected.

8. The chemiluminescence immunoassay kit according to claim 4 or 6, wherein: The competitive small molecule and the small molecule to be detected can be recognized and bound by the same antibody coated on the surface of the luminescent microparticles; preferably, the binding forces of the competitive small molecule and the small molecule to be detected with the antibody are different; more preferably, the binding force of the competitive small molecule with the antibody is lower than the binding force of the small molecule to be detected with the antibody.

9. The chemiluminescence immunoassay kit according to claim 4 or 6, wherein: It further includes reagent 3, and reagent 3 includes a releasing agent, and the releasing agent is a strong acid, a strong base and / or a displacing agent; preferably, the releasing agent is a citrate buffer solution; and / or It further includes reagent 4, and reagent 4 includes avidin-coated photosensitive microparticles; preferably, the concentration of reagent 4 is 40 μg / mL - 50 μg / mL.

10. Use of a chemiluminescence immunoassay kit as described in any one of claims 4 - 9 for detecting a small molecule to be detected; preferably, the molecular weight of the small molecule to be detected is less than 1000 Da.

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